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基本信息

質粒圖譜和多克隆位點信息 

注意：

客戶在我公司選擇構建載體服務項目時，請自行提供母源載體。

如要使用本公司保存的母源載體時，本公司不能保證所有母源載體完全符合客戶預期，也不承擔由此產生的任何責任。

載體描述

pDsRed-Monomer-N1載體是熒光報告載體，表達C端DsRed-Monomer融合蛋白；DsRed-Monomer是DsRed的單體突變體，與DsRed相比，編碼序列經過了人工優化，適用于在哺乳動物細胞中的高水平表達。

pDsRed-Monomer-N1 is a mammalian expression vector that encodes DsRed-Monomer (DsRed.M1), a monomeric mutant derived from the tetrameric Discosoma sp. red fluorescent protein DsRed. DsRed-Monomer contains forty-five amino acid substitutions. When DsRed-Monomer is expressed in mammalian cell cultures, red fluorescent cells can be detected by either fluorescence microscopy or flow cytometry 12–16 hr after transfection (DsRed-Monomer excitation and emission maxima = 557 nm and 592 nm, respectively). The DsRed-Monomer coding sequence is human codon-optimized for high expression in mammalian cells.

DsRed-Monomer is well suited for use as a fusion tag. The multiple cloning site (MCS) in pDsRed-Monomer-N1 is positioned between the immediate early promoter of CMV (PCMV IE) and the DsRed-Monomer coding sequence. Genes cloned into the MCS are expressed as fusions to the N-terminus of DsRed-Monomer if they are in the same reading frame as DsRed- Monomer and there are no intervening stop codons. A Kozak consensus sequence is located immediately upstream of the DsRed-Monomer gene to enhance translational efficiency in eukaryotic systems. SV40 polyadenylation signals downstream of the DsRed-Monomer gene direct proper processing of the 3' end of the DsRed-Monomer mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin-resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists of the SV40 early promoter, the neomycin/ kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter upstream of the cassette confers kanamycin resistance in E. coli.

應用

pDsRed-Monomer-N1 can be used to construct fusions to the N-terminus of DsRed-Monomer. If a fusion construct retains the fluorescent properties of the native DsRed-Monomer protein, its expression can be monitored by flow cytometry and its localization in vivo can be determined by fluorescence microscopy. The target gene must be cloned into pDsRed-Monomer-N1 so that it is in frame with the DsRed-Monomer coding sequence, with no intervening in-frame stop codons. The inserted gene must include an initiating ATG codon. Recombinant pDsRed-Monomer-N1 can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418. pDsRed-Monomer-N1 can also be used as a cotransfection marker; the unmodified vector will express DsRed-Monomer.

The DsRed1-N Sequencing Primer (Cat. No. 632387) can be used to sequence genes cloned adjacent to the 5' end of the DsRed-Monomer coding region.

For Western blotting, the Living Colors? DsRed Polyclonal Antibody (Cat. No. 632496) can be used to recognize the DsRed-Monomer protein. However, to generate optimal results it may be necessary to use a higher concentration of antibody than recommended on the DsRed Polyclonal Antibody Certificate of Analysis.

載體序列

LOCUS       Exported                4691 bp ds-DNA     circular SYN 20-MAR-2019

DEFINITION  synthetic circular DNA

ACCESSION   .

VERSION     .

KEYWORDS    .

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 4691)

  AUTHORS   BT-Lab

  TITLE     Direct Submission

  JOURNAL   Exported Wednesday, Mar 20, 2019 from SnapGene Viewer 4.3.4

FEATURES             Location/Qualifiers

     source          1..4691

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     enhancer        61..364

                     /label=CMV enhancer

                     /note="human cytomegalovirus immediate early enhancer"

     promoter        365..568

                     /label=CMV promoter

                     /note="human cytomegalovirus (CMV) immediate early

                     promoter"

     misc_feature    591..671

                     /label=MCS

                     /note="multiple cloning site"

     regulatory      673..682

                     /regulatory_class="other"

                     /note="vertebrate consensus sequence for strong initiation

                     of translation (Kozak, 1987)"

     CDS             679..1356

                     /codon_start=1

                     /product="monomeric derivative of DsRed fluorescent protein

                     (Strongin et al., 2007)"

                     /label=DsRed-Monomer

                     /note="mammalian codon-optimized"

                     /translation="MDNTEDVIKEFMQFKVRMEGSVNGHYFEIEGEGEGKPYEGTQTAK

                     LQVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYMKLSFPEGFTWERSMNFEDGGV

                     VEVQQDSSLQDGTFIYKVKFKGVNFPADGPVMQKKTAGWEPSTEKLYPQDGVLKGEISH

                     ALKLKDGGHYTCDFKTVYKAKKPVQLPGNHYVDSKLDITNHNEDYTVVEQYEHAEARHS

                     GSQ"

     polyA_signal    1479..1600

                     /label=SV40 poly(A) signal

                     /note="SV40 polyadenylation signal"

     rep_origin      complement(1607..2062)

                     /direction=LEFT

                     /label=f1 ori

                     /note="f1 bacteriophage origin of replication; arrow

                     indicates direction of (+) strand synthesis"

     promoter        2089..2193

                     /gene="bla"

                     /label=AmpR promoter

     promoter        2195..2552

                     /label=SV40 promoter

                     /note="SV40 enhancer and early promoter"

     rep_origin      2403..2538

                     /label=SV40 ori

                     /note="SV40 origin of replication"

     CDS             2587..3381

                     /codon_start=1

                     /gene="aph(3')-II (or nptII)"

                     /product="aminoglycoside phosphotransferase from Tn5"

                     /label=NeoR/KanR

                     /note="confers resistance to neomycin, kanamycin, and G418

                     (Geneticin(R))"

                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP

                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS

                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ

                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA

                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"

     polyA_signal    3613..3660

                     /label=HSV TK poly(A) signal

                     /note="herpes simplex virus thymidine kinase

                     polyadenylation signal (Cole and Stacy, 1985)"

     rep_origin      3989..4577

                     /direction=RIGHT

                     /label=ori

                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of

                     replication"

ORIGIN

        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg

       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt

      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca

      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc

      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta

      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac

      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg

      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg

      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt

      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta

      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg

      661 gatccaccgg tcgccaccat ggacaacacc gaggacgtca tcaaggagtt catgcagttc

      721 aaggtgcgca tggagggctc cgtgaacggc cactacttcg agatcgaggg cgagggcgag

      781 ggcaagccct acgagggcac ccagaccgcc aagctgcagg tgaccaaggg cggccccctg

      841 cccttcgcct gggacatcct gtccccccag ttccagtacg gctccaaggc ctacgtgaag

      901 caccccgccg acatccccga ctacatgaag ctgtccttcc ccgagggctt cacctgggag

      961 cgctccatga acttcgagga cggcggcgtg gtggaggtgc agcaggactc ctccctgcag

     1021 gacggcacct tcatctacaa ggtgaagttc aagggcgtga acttccccgc cgacggcccc

     1081 gtaatgcaga agaagactgc cggctgggag ccctccaccg agaagctgta cccccaggac

     1141 ggcgtgctga agggcgagat ctcccacgcc ctgaagctga aggacggcgg ccactacacc

     1201 tgcgacttca agaccgtgta caaggccaag aagcccgtgc agctgcccgg caaccactac

     1261 gtggactcca agctggacat caccaaccac aacgaggact acaccgtggt ggagcagtac

     1321 gagcacgccg aggcccgcca ctccggctcc cagtagagcg gccgcgactc tagatcataa

     1381 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc

     1441 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata

     1501 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc

     1561 attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt

     1621 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag

     1681 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt

     1741 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga

     1801 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg

     1861 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct

     1921 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc

     1981 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt

     2041 aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct

     2101 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga

     2161 taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga

     2221 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa

     2281 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca

     2341 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc

     2401 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt

     2461 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag

     2521 gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg

     2581 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg

     2641 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg

     2701 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat

     2761 gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca

     2821 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg

     2881 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat

     2941 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa

     3001 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg

     3061 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg

     3121 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg

     3181 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat

     3241 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac

     3301 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc

     3361 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc

     3421 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg

     3481 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt

     3541 tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa

     3601 cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc

     3661 ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt

     3721 ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag

     3781 gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat

     3841 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc

     3901 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag

     3961 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct

     4021 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac

     4081 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc

     4141 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg

     4201 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt

     4261 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt

     4321 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc

     4381 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca

     4441 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata

     4501 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg

     4561 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct

     4621 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta

     4681 ccgccatgca t

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